DYNAMIC ASPECTS — AMINO ACID POOL TURNOVER 655 
DISCUSSION 
Chairman: JOHN REINER 
REINER: I would like to exercise the privilege of the Chair and ask Dr. Cowte about this suggested 
set of tunnels here. You are assuming, as I understand it, that up to a point, namely the point at 
which the final amino acid is synthesized, all the reactions are really the same in all the tunnels. 
Are you taking care of the fact that things go just the right way by implying that some mecha- 
nism picks off the final product just as soon as it is formed, or are you proposing that a varying 
number of terminal enzymes are missing in the various tunnels? 
Cowie: Every attempt to determine the state of the amino acids in the internal pool during 
their transfer to protein has been negative. We have, as I have indicated, specifically labeled 
the internal pool and tried, without success, to interfere with its transfer to protein by adding a 
large quantity of exogenous material. Furthermore, amino acid analogs, once in the internal 
pool show no final selection steps for protein incorporation even though discrete selection steps 
could be observed prior to internal pool incorporation of these analogues. One interpietation of 
the model presented is that the amino acids in the internal pool (or in the “tunnels”) are already 
on the final sites awaiting appropriate conversion and final peptide linkage. 
HENDLER: Dr. Cowle, could you give me some idea of your thoughts on the type of bonds which 
may be involved in holding your internal pool in terms, for example, of the type of washing pro- 
cedures which leave it intact or remove it? 
Cowie: One of the distinctions between the two kinds of amino acid pools is the following: 
hot water or 5 per cent cold TCA will extract both pools together; cold water will extract only the 
expandable pool, leaving intact the internal pool. We have no knowledge of what kind of affinity 
or association these amino acids form to account for this distinction or how the internal pool 
material is adsorbed. Perhaps VAN DER WAAL forces or hydrogen binding might be the answer. 
A. Miter: I would like to ask Dr. Cowle if he has considered models that go beyond the 
macromolecular stage. There is a great deal of fine structure in an organism such as Candida. 
Cowte: I think I am in enough trouble already, but since you ask, I would like to suggest that 
the pool might be associated sed the cellular ribosomes; that the internal pool is actually com- 
plexed with the protein-forming system, and that the ribosomes are indeed this system. 
A. MILLER: I was wondering if you had considered models in which the external amino acids enter 
vacuoles rather than mix with the cell contents. 
Cowie: We have looked into all kinds of partitioning systems in which the amino acids of the 
internal pool are in bags or sacks and even in vacuoles. When you do this, however, these sacks 
or vacuoles must be able to account for all the experimental data available. The concentrating 
mechanism, the conversion system, and the final transfer from the bag or sack to protein (without 
competition) must be accounted for. It would be a pleasure for me if someone would use the bag 
o1 vacuole system and construct a satisfactory model taking into account all the information on 
the pools that has accumulated. So far, a bag system has not been postulated which reasonably 
accounts for the kinetic and other experimental evidence observed. 
A. MILLER: I would like to ask Dr. HALvorson if he has any data on recovery from the in- 
creased turnover during starvation. How does the cell get rid of all these lytic enzymes? 
Hatvorson: I really have no information as to the level of the proteolytic activity on reinocula- 
tion into fresh media. In our yeast experiments we have tried to make measurements of the 
proteolytic activity as a function of growth; that is, the A proteolytic activity/ A mass. We found 
that this ratio dramatically increases as the cell begins to decrease its growth rate, so that you 
get what looks like a preferential synthesis but w hich we expect is a release of enzyme from the 
particulate fraction. When the cells are put into fresh growth media, this ratio again reverts to 
normal. In the experiments of SYLVEN ef al. one wonders whether the proteolytic systems them- 
selves are subject to autodegradation. Another possibility is that the proteolytic enzymes are 
masked in some way when functional particles are reformed. 
We have had asimilar problem in examining de-adaptation in yeast where, upon removal of the 
inducer, a net loss of the enzyme occurred. The question of whether this enzyme is being degraded 
or inactivated is difficult to answer. We have studied this protein using specific antisera against 
